Enzymes

Lab Section 2.4

Enzymes

• Protein catalysts• Have complex 3-D

structures• Pockets act as active sites

– catalyze specific chemical reactions

E + S E-S Complex E + P

Factors Affecting Enzyme Activity

• Concentration of Enzyme• Concentration of Substrate• Concentration of

Cofactors / Coenzymes• Temperature• pH

Measuring Enzyme Concentration Using Beer’s Law

• Measure [enzyme] indirectly via reaction rates– Enzyme activity (reaction rate)

[enzyme]

• During the reaction, substrate → product– Rate = how much product is

formed per unit time

– So, [Enzyme] α Δ[Product]/time

Measuring Enzyme Concentration Using Beer’s Law

• If product formed absorbs light…– [product] α A

• As [product] changes, A changes proportionally

• Since [product]/time α A/time and [product]/time α [enzyme]…

• Therefore: A/time α [enzyme]

International Units (U)

• Measurement of enzymatic activity (U)– Quantity of enzyme needed to convert 1 mol

substrate into product in 1 minute

• Measure of enzymatic function, indirectly enzyme concentration

• Often expressed as concentration (U/L)• Determined by examining [product] / min

Alkaline Phosphatase (ALP)

• Phosphatase– Enzyme that removes phosphate groups from proteins– Normally low levels of ALP in blood plasma– Elevated levels indicate pathology

• Liver and bone disease, Hodgkin’s disease, congestive heart failure, hyperparathyroidism, intestinal disease, etc.

• Enzyme activity measured by reacting with substrate to form light-absorbing product– p-nitrophenol phosphate + H2O

p-nitrophenol + hydrogen phosphate

Procedures

• Timing is critical once the reaction has begun!– Have your spec already zeroed with the blank before you

start the reaction.

• use 2 samples:– BLANK (3.0 ml of reagent and 0.1 ml distilled water)

– BLOOD (3.0 ml of reagent and 0.1 ml serum)

• Place in test tube in 30 C bath for 1 min

Procedures

• Remove tube, dry outside, place in spec, and read absorbance

• Replace in water bath

• Remove, dry and read absorbance at 2 min

• Repeat to measure absorbance at 3 min, 4 min, and 5 min

Determining ALP Activity

Determine average change in absorbance per minute

(A5min – A1min)/4 = A/min Determine alkaline phosphatase activity

 A/min x TV (ml) x 1000 (ml/L) = activity (U/L)

18.45 x LP x SV (ml)